Abstract

The impermeability of the mitochondrial inner membrane to the chelator ethanedioxybis(ethylamine)tetra-acetic acid permits discrimination between Ca2+which has been transported to the internal (matrix) phase and Ca2+which binds to the external surfaces of the mitochondrion. With this technique, it is shown that ‘energy-independent high-affinity’ binding is a measure of carrier-mediated active Ca2+transport in respiration-inhibited mitochondria; the carrier also transports Ca2+to the internal phase after treatment with carbonyl cyanide m-chlorophenylhydrazone, but in this case the active-transport component is inhibited. The Ca2+-binding sites associated with the external membrane surfaces are similar in concentration and affinity for both inhibited and uncoupled mitochondria; it was not possible to measure external Ca2+binding which could be identified as carrier specific. The results are discussed in relation to the mechanism of mitochondrial Ca2+transport, and to previous studies of energy-independent Ca2+binding.